1. Field of the Invention
The present invention relates to TCF mutants comprising a novel amino acid sequence, more specifically, TCF mutants which are obtained by mutagenesis of one or more amino acid in the sequence from N-terminus to the first kringle of native TCF and show lowered affinity to heparin and/or elevated biological activity. The TCF mutants of the present invention which show proliferative activity and growth stimulative activity in hepatocyte are beneficial for treatment of various hepatic diseases and as an antitumor agent.
2. Background Art
Tumor cytotoxic factor (TCF-II) produced in human fibroblast cells is a novel antitumor substance different from any antitumor proteins so far reported. The present inventors have succeeded in the cloning of cDNA coding for the protein of the present invention, determined the total amino acid sequence thereof and confirmed usefulness thereof (WO90/10651). The molecular weight of TCF was 78,000xc2x12,000, or 74,000xc2x12,000 according to the results of SDS electrophoresis under non-reducing conditions, while the results under reducing conditions indicated A-chain of 52,000xc2x12,000, common band, B-chain of 30,000xc2x12,000 and/or C-chain of 26,000xc2x12,000. TCF is a protein which has a high affinity to heparin or heparin-like substance and shows high antitumor activity against tumor cells and proliferative activity to normal cells. Further, it was confirmed that it belongs to a wide variety of family of HGF, a growth factor for hepatocyte. Therefore, since TCF is not only an antitumor factor, but also a growth factor for hepatocytes, it is known that it is beneficial for liver regeneration after hepatectomy. Much research been carried out from the aspects of structure-function relationship of hepatocyte growth factor(HGF) so far. About 20 species of deletion mutants and about 50 species of point mutants have been reported so far (K. Matsumoto, et. al., Biochem. Biophys. Res. Comm., vol. 181, pp 691-699 (1991); G. Hartmann, et. al. Proc. Natl. Acad. Sci. USA, vol. 89, pp11574-11587 (1992); N. A. Lokker, et. al., EMBO J. vol. 11, pp 2503-2510 (1992); M. Okigaki et. al., Biochemistry, vol. 31, pp 9555-9561 (1992); N. A. Locker, et. al. Protein Engineering, vol. 7, pp895-903 (1994)), however, any mutant which clearly shows an elevated biological activity has not been obtained at present. Half-life of TCF in vivo is known to be extremely short, about 2 minutes. Therefore, it is anticipated that a comparatively large amount of the protein should be administered for treatment of various diseases. It is conceivable that the dosage level of TCF administered will be reduced by elevation of biological activity thereof or by prolongation of the half-life thereof in vivo. Though it was described on TCF mutants with prolonged half-life in patent publication W094/14845, any TCF mutant with elevated biological activity has not been obtained at present, like HGF described above.
Therefore, the present inventors have conducted an investigation to obtain a TCF mutant which shows elevated biological activity or prolongation of half-life in vivo. More specifically, the present inventors have carried out research to obtain the above-mentioned mutant with elevated biological activity or with prolonged half-life in vivo which is different from native TCF with respect to amino acid sequence by altering the DNA sequence coding for the amino acid sequence of native TCF and expressing DNA thereof. Accordingly, an object of the present invention is to provide a TCF mutant with elevated biological activity or with prolonged half-life in vivo due to lowered affinity to heparin.
The present inventors have eagerly investigated the above and obtained novel TCF mutants which have amino acid sequences different from that of TCF mutant found prior to the present invention and show elevated biological-activity and/or lowered affinity to heparin. The present invention provides TCF mutants which show more than 10 folds of specific activity (biological activity per unit amount of protein) and/or lowered affinity to heparin.
These are the first mutants with extremely elevated biological activity by mutagenizing the amino acid sequence of native TCF.
An object of the present invention is to provide a TCF mutant with lowered affinity to heparin and/or with elevated biological activity which is obtained by mutagenesis of one or more amino acid residue(s) in the amino acid sequence from N-terminus to the first kringle of native TCF.